All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
A wide variety of antibacterial and antifungal proteins have been isolated from both animals and plants. Because of the major differences in the structures of fungal, gram positive bacterial and gram negative bacterial cell walls, many of these proteins attack only fungi, gram positive or gram negative bacteria. Due to the very different natures of the outer cell walls of these microbes (see FIG. 1A-1C), those antimicrobial proteins that kill fungi and gram positive bacteria tend to be enzymes that can depolymerize the exposed cell walls of these microbes; those that attack gram negative bacteria tend to destabilize and permeabilize the exposed cell membrane.
During the last two decades, literally hundreds of antimicrobial proteins and peptides (proteins with less than 50 amino acids) have been discovered in plants and in the cells and body fluids of multicellular animals from mollusks to humans. Some antimicrobial peptides are always present in the host, while others are induced in response to infection or inflammation (Jaynes et al 1987; Mitra and Zhang 1994; Broekaert et al 1997; Nakajima et al 1997; Vunnam et al 1997). Among the most well described antimicrobial proteins are peptides with broad spectrum activity against bacteria, fungi, enveloped viruses, parasites, and tumor cells (Hancock and Lehrer 1998). More than 500 such peptides have been found in diverse organisms.
Antimicrobial peptides vary greatly in length and primary structure, but a common feature is that they are amphiphathic and cationic (Andreu and Rivas 1998; Gacia-Olmedo et al 1998; Nissen-Meyer and Nes 1997). Antimicrobial peptides have a cationic charge at physiological pH because of an excess of lysine and arginine residues and they have approximately 50% hydrophobic amino acids. This charge facilitates electrostatic attraction to negatively charged surfaces of a wide range of microbes. Their ability to assume amphipathic structures allows direct interaction with ubiquitous phosphoglycerol-lipids and incorporate into microbial membranes, resulting in membrane depolarization, electrolyte leakage, and lysis. Antimicrobial peptides can be categorized as either linear peptides (e.g. cecropins, attacins and magainins) or disulfide-linked peptides (e.g. defensins, prophenins and thaumatins).
Antimicrobial Linear Peptides.
Cecropins and melittins belong to the most abundant class of linear antimicrobial peptides. Both form α-helices in solution. Cecropins were first isolated from the hemolymph of Hyalophora cecropia, the giant silk-moth, and very similar molecules have since been isolated from other insects. Cecropins are cylindrical, amphipathic molecules with long hydrophobic regions on one end. Cecropins cause leaky cell membranes and can lyse bacterial and fungal cells; in effect, acting like detergents. Linear peptides are not found naturally in plants. Although highly effective in killing bacteria in petri dishes, linear peptides are usually rapidly degraded by plant proteases, and are therefore much less effective in plata. Transgenic tobacco plants expressing cecropins have slightly increased resistance to Pseudomanas syringae pv. tabaci, the cause of tobacco wildfire (Huang et al 1997). Cecropin residues crucial for lethality have been well-defined, and shortened synthetic analogs also exhibit antifungal activity. Synthetic cecropin analogs Shiva-1 and SB-37, expressed from transgenes in potato plants, reduced bacterial infection caused by Erwinia carotovora (Arce et al 1999). Transgenic apple expressing the SB-37 peptide analog showed increased resistance to E. amylovora in field tests (Norelli et al 1998). However, several researchers have reported that the antimicrobial protein cecropin B is rapidly degraded when incubated with intercellular fluid, with a half-life in intercellular fluids ranging from about three minutes in potato to about 25 hours in rice (Owens & Heutte, 1997).
Synthetic cecropins also appear to suffer from proteolytic degradation by plants. Melittin is the principal toxic component in the venom of the European honeybee (Apis mellifera) and by contrast with cecropins, is highly hemolytic and also phytotoxic. Using molecular modeling and genetic engineering, the melittin residues involved in toxicity were identified and replaced by those from the structurally related cecropin peptide. The resulting chimeric gene MsrAl showed reduced hemolytic activity and phytotoxicity but retained its broad-spectrum antimicrobial properties (Osusky et al 2000). When MsrAl is expressed in transgenic potatoes, the potato tubers show resistance to the fungal pathogens Phytophthora cactorum and Fusarium solani and the bacterium E. carotovora. 
Attacins form another group of linear antibacterial proteins that are considerably larger than cecropins (>180 amino acids). The mechanism of antibacterial activity of these proteins is to inhibit the synthesis of outer membrane proteins in gram negative bacteria. Transgenic potatoes expressing the attacin gene showed resistance to bacterial infection by Erwinia carotovora (Arce et al 1999). Transgenic pear and apple expressing attacin genes have also shown enhanced resistance to E. amylovora (Norelli et al 1994; Reynoird et al 1999; Ko et al 2000). Ko and coworkers engineered transgenic apple plants using the attacin E gene, both with and without a signal peptide to transport the attacin into the intercellular space. They found that transgenic plants with attacin fused to a signal peptide had better disease resistance than plants carrying attacin without the signal peptide, even though the plants with the signal peptide had a much lower attacin content than lines without a signal peptide. Attacin E was also found to be rapidly degraded by plants.
Magainins are a third group of linear antimicrobial peptides, 22-24 amino acids in length, originally isolated from frog skin (Li et al 2001). The mode of action of these peptides involves the disruption of microbial cell membranes. They show strong inhibitory activity against a variety of bacteria and fungi in vitro, including many plant pathogens, but as with all linear peptides, are also highly susceptible to plant proteases. Li et al (2001) tested a synthetic magainin analog, Myp30, that had been modified to be less sensitive to extracellular plant proteases. Transgenic tobacco plants expressing Myp30 were somewhat resistant to the fungal pathogen Peronospora tabacina and the bacterial pathogen E. carotovora. 
Searches for shorter, more potent antimicrobial peptides have led to the development of entirely synthetic peptides and also synthetic derivatives of natural peptides with broader and higher antimicrobial activity than their natural counterparts. Cary et al (2000) reported that the expression of the 17 amino acid synthetic peptide D4E1 in transgenic tobacco gave resistance to several fungal and bacterial pathogens. Ali and Reddy (2000) tested four synthetic peptides for their ability to inhibit growth of important plant pathogens in vitro and in detached potato leaf and tuber assays. Fungal growth was inhibited by all four peptides, while growth of two Erwinia species was inhibited by two of the peptides.
Antimicrobial, Disulfide-Linked Peptides.
Lysozymes are enzymes that hydrolyze the peptidoglycan layer of the bacterial cell wall. Hen egg-white lysozyme, bacteriophage T4 lysozyme, and human lysozyme genes have been cloned and transferred to several plant species in attempts to enhance bacterial or fungal disease resistance. Hen egg-white lysozyme genes have been used to confer bacterial disease resistance to transgenic tobacco plants (Trudel et al 1995; Kato et al 1998). Bacteriophage T4 lysozyme has also been reported to enhance resistance in transgenic potato against the bacterial soft rot pathogen E. carotovora (During et al 1993; Ahrenholz et al., 2000) and in transgenic apple plants against the bacterial fire blight pathogen E. amylovora fireblight infection (Ko 1999). Human lysozyme transgenes have conferred disease resistance in tobacco through inhibition of fungal and bacterial growth, suggesting the possible use of the human lysozyme gene for controlling plant disease (Nakajima et al 1997). However, lysozymes can be skin irritants, and have the potential to become allergenic.
Thanatin is a 21-residue inducible peptide found in the hemipteran insect Podisus maculiventris. Thanatin exhibits the broadest range of antimicrobial activity so far characterized (Taguchi et al 2000). Unfortunately, thanatin exhibits powerful cytotoxic effects on many eukaryotic cell types, making them unsuitable for any therapeutic use as antibiotics and also likely unsuitable for use in plants to control pathogens.
Plant, mammalian and insect defensins belong to the class of antimicrobial peptides characterized by β-sheet structures. These complex folded molecules contain four, six, or eight invariant cysteine residues that form several intramolecular disulfide bonds. The active peptides have antibacterial, antifungal, and antiviral activities. Defensins display antimicrobial activity through binding and disruption of microbial plasma membranes. A 5.6 kDa antifungal peptide alfAFP was isolated from alfalfa seeds (Medicago sativa) (Guo et al 2000). Expression of the peptide in transgenic potato plants gave robust resistance to the fungal pathogen Verticiliuin dahliae. The construct was not tested against bacteria.
Prophenins belong to a new class of antimicrobial peptides first discovered in mammalian white blood cells (Wang et al 1999). Prophenins display exceptionally strong endotoxin (LPS) binding activity as well as antimicrobial activity, even after proteolytic degradation. They are stabilized by two disulfide bonds. These peptides show promise as a potent new class of antibiotics for gram-negative bacterial infections in animals.
Thaumatin and thaumatin-like proteins are made by plants and constitute one of five major classes of PR proteins that are characterized by a sweet taste (to humans), small size (22 kDa) and most importantly, a highly stabilized, compact structure with eight disulfide bonds that renders them very resistant to protease degradation (Selitrennikoff, 2001).
Antimicrobial Bacteriophage Proteins.
All bacteriophages must escape from bacterial host cells, either by extrusion from the host cell, as with filamentous phages, or by host cell lysis. Host cell lysis requires two events: ability to penetrate the inner membrane of both gram negative and gram positive bacteria (see FIGS. 1A and 1B), and ability to depolymerize the murein layer, which is relatively thick in gram positive cell walls.
Penetration of the inner membrane is accomplished in many, but evidently not all, phage by use of small membrane-localized proteins called “holins” that appear to accumulate in the bacterial inner membrane until reaching a specific concentration, at which time they are thought to self-assemble to permeabilize the inner membrane (Grundling et al., 2001; Wang et al. 2000; Young et al., 2000). The terms “holin” and “holin-like” are not biochemically or even functionally accurate terms, but instead as used herein refer to any phage protein capable of permeabilizing the inner membrane, thereby allowing molecules other than holins that are normally sequestered in the cyctoplasm by the inner membrane, including proteins such as endolysins, to breach or penetrate the bacterial cell membrane barrier to reach the cell wall. Holins are sometimes found with “accessory proteins” of unknown function. The biochemical function(s) of holins is speculative; most, if not all of the current knowledge on holins is based on the λ phage S protein (Haro et al. 2003).
Holins are encoded by genes in at least 35 different families, having at least three topological classes (classes I, II, and III, with three, two and one transmembrane domains [TMD], respectively), all with no detected orthologous relationships (Grundling et al., 2001). At least two holins are known to be hemolytic and this hemolytic function has been hypothesized to play a role in the pathogenesis of certain bacteria towards insects and nematodes (Brillard et al., 2003). Only a few have been partially characterized in terms of in vivo function, leading to at least two very different theories of how they may function. Indeed, no holin genes have been found or suggested in many phage, despite the ready availability of genomic sequence data. The most widely accepted theory is that holins function to form oligomeric membrane pores (Graschopf & Blasi, 1999; Young et al., 2000); most of the supporting data is based upon studies of the holin of phage B. A second theory is that holins form an oligomeric “raft” in the membrane that constitutes a lesion (Wang et al., 2003). Both theories may be correct for different holins, and other holins may perform their functions in very different manners.
Depolymerization of the murein layer is accomplished by lytic enzymes called endolysins. There are at least three functionally distinct classes of endolysins: 1) glucosaminidases (lysozymes) that attack the glycosidic linkages between the amino sugars of the peptidoglycan; 2) amidases that attack the N-acetylmuramoyl-L-alanine amide linkage between the glycan strand and the cross-linking peptide, and 3) endopeptidases that attack the interpeptide bridge linkages (Sheehan et al., 1997). Endolysins are synthesized without an export signal sequence that would permit them access to the peptidoglycan (murein) layer, and they therefore usually accumulate in the cytoplasm of phage infected bacteria until they are released by the activity of holins (Young and Blasi, 1995).
Lysozymes have been suggested as useful antibiotics that can be used as external agents against both Gram-positive and Gram-negative bacteria because at least some of them are multifunctional (During et al., 1999). This dual functionality is based on the finding that both phage T4 and hen egg white lysozyme have both glucosaminidase activity as well as amphipathic helical stretches that allow them to penetrate and disrupt bacterial, fungal and plant membranes (During et al., 1999). The microbicidal activity of lysozymes can be affected by C-terminal additions; additions of hydrophobic amino acids decreased activity against gram positive, but increased activity against E. coli (Arima et al., 1997; Ito et al., 1997). Additions of histidine, a hydrophilic amino acid, to T4 lysozyme doubled its antimicrobial activity against Gram-positive and Gram-negative bacteria (During et al., 1999). The nonenzymatic, microbicidal function of lysozymes appeared to be due to amphipathic C-terminal domains that could be mimicked by small synthetic peptides modeled after the C-terminal lysozyme domains (During et al., 1999). As described above, transgenic plants have been created that express lysozymes and give some resistance to certain plant pathogens. Since most endolysins accumulate to high titers within the bacterial cell without causing lysis, endolysins other than certain lysozymes such as T4 would not be expected to attack Gram-negative bacteria if externally applied, since Gram-negative bacteria are surrounded with an outer membrane comprised of a lipid bilayer that would protect its murein layer from enzymatic attack just as effectively as its inner membrane does. Also as mentioned earlier, lysozymes are also skin irritants, probably as a result of their ability to invade membranes.
Attempts have been made to treat bacterial diseases of both animals and plants by use of intact bacteriophage. All of these attempts have severe limitations in their utility. For examples, U.S. Pat. No. 5,688,501 discloses a method for treating an infectious disease of animals using intact bacteriophage specific for the bacterial causal agent of that disease. U.S. Pat. No. 4,957,686 discloses a method for preventing dental caries by using intact bacteriophage specific for the bacterial causal agent of dental caries. Flaherty et al. (2000) describe a method for treating an infectious disease of plants using intact bacteriophage specific for the bacterial causal agent of that disease. In all these cases and in similar cases using intact bacteriophage, the bacteriophage must attach to the bacterial host, and that attachment is highly host specific, limiting the utility of the phage to specific bacterial host species, and sometimes specific bacterial host strains. In addition, for attachment to occur, the bacteria must be in the right growth phase, and the phage must be able to gain access to the bacteria, which are often buried deep within tissues of either animals or plants, or shielded by bacterial biofilms.
Attempts have been made to treat gram-positive bacterial diseases of animals, but not plants, by use of lytic enzyme preparations extracted from bacteriophage infected bacteria or from bacteria expressing bacteriophage genes. These, too, have serious limitations. For example, U.S. Pat. No. 5,985,271 discloses a method of treating an animal disease caused by a specific gram positive bacterium, Streptococcus, by use of a crude specific endolysin preparation. Similarly, U.S. Pat. No. 6,017,528 discloses a method of preventing and treating Streptococcus infection of animals by use of a crude specific endolysin preparation. Similarly, WO 01/90331 and US 2002/0058027 disclose methods of preventing and treating Streptococcus infection of animals by use of a purified preparation consisting of a specific endolysin. In all of these cases, the enzyme preparations must be purified, buffered, prepared for delivery to the target areas and preserved at the target site. In addition, the enzyme must be able to gain access to the infecting bacteria, and be present in sufficient quantity to kill the growing bacteria. None of these methods would be useful in the treatment of gram negative bacteria, because the endolysins could not penetrate the outer membrane of such bacteria. Attempts have been made to treat both gram-positive and gram-negative bacterial diseases of animals, but not plants, by use of lytic enzyme preparations extracted from bacteriophage infected bacteria or from bacteria expressing bacteriophage genes. WO 01/51073, WO 01/82945, WO 01/019385, US 2002/0187136 and US 2002/0127215 disclose methods of preventing and treating a variety of gram positive and gram negative bacterial infections of animals by use of lytic enzymes that may optionally include specific “holin lytic enzymes” or “holin enzymes”.
Since holins are not known to exhibit enzymatic function, and since examples of such holin lytic enzymes are not demonstrated or taught in WO 01/51073, WO 01/82945, WO 01/19385, US 2002/0187136 and US 2002/0127215, such enzymes appear to represent a theoretical and undemonstrated enzyme defined by reference to a desirable characteristic or property. As correctly stated elsewhere by the same inventors: “Holin has no enzymatic activity” (refer WO 01/90331, page 9 line 12). Lytic enzymes, which form the basis for the methods disclosed in all of these PCT publications, are internally defined: “The present invention is based upon the discovery that phage lytic enzymes specific for bacteria infected with a specific phage can effectively and efficiently break down the cell wall of the bacterium in question. At the same time, the substrate for the enzyme is not present in mammalian tissues . . . ” (WO 01/51073 paragraph 3, page 4). “The lytic enzymes produced by bacterial phages are specific and effective for killing select bacteria.” (paragraph 2, page 7).
The term “holin enzyme” as used in Claim #3 of WO 01/51073 refers to the enzymes defined in Claim #1 as “the group consisting of lytic enzymes, modified lytic enzymes and combinations thereof . . . ” Similar references in the claims of WO 01/82945, WO 01/019385 and US 2002/0187136 and US 2002/0127215 may be found. None of these patent applications disclose or claim the use of holin or holins alone, without enzymatic activity, in any manner, including the formulation of a compound or method of treatment of animal or plant diseases. Indeed, absent the teachings of the present invention, one skilled in the art would not have expected holins without lytic activity to kill gram positive bacteria, since holins without lytic activity would not be able to penetrate the thick gram positive bacterial cell wall.
WO 02/102405 discloses a method of preventing food poisoning in animals by inclusion of a purified preparation consisting of specific lytic enzymes and optionally, specific lytic “holin enzymes”. Again, since holins are not known to exhibit enzymatic function, it is unclear as to what is taught or specified in the claims, other than a theoretical and undemonstrated enzyme defined by reference to a desirable characteristic or property.
In all previously published cases wherein holins are incorporated, used or described, enzyme preparations are involved. These enzyme preparations must be purified, buffered, prepared for delivery to the target areas and preserved at the target site.
Thus, the prior art reviewed herein fails to teach the use of holins, holin genes or of chimeric holins, without enzyme activity, for the control of bacterial or fungal diseases and pests. This prior art also fails to teach the use of holins combined with endolysin genes in the formulation of a compound or method of treatment of plant diseases. Furthermore, this prior art fails to teach the use of holin genes or modified holin genes or of chimeric holin and endolysin genes in the creation of transgenic animals or plants capable of fighting diseases.
It has been suggested that a specific endolysin from a bacteriophage that attacks a gram negative bacterial plant pathogen might be effective in providing resistance to that pathogen if the endolysin gene were cloned and expressed in plants (Ozawa et al., 2001). This suggestion is most unlikely, since endolysins other than T4 lysozyme are not known to penetrate bacterial membranes, and Gram-negative bacteria have a distinctive outer membrane that provides a strong environmental barrier that is impermeable to most molecules.
As described elsewhere herein, the present invention provides membrane destabilization and permeabilization based upon the action of unique bacteriophage proteins called holins. The present invention is based, in part, on our discovery that holins not only destabilize and permeabilize bacterial inner membranes from inside bacterial cells, but in addition, work externally as well, presumably destabilizing and permeabilizing outer membranes as well as inner membranes. Activity of holins in destabilization and permeabilization of the outer membrane presumably allows natural defense molecules secreted by plants and/or by other microbes to breach the outer membrane of the target cells, thereby compromising the “barrier function” of the outer membrane. Kingsley et al., (1993) provide strong evidence that the outer membrane of a plant pathogenic bacterium can function as a barrier in preventing plant defense molecules from the killing the bacteria. Target cells can be bacterial, fungal, insect or nematode. The invention also provides the incorporation of enzymatic cell wall depolymerization based upon unique bacteriophage proteins called endolysins and provides the incorporation of both holin and endolysin function in a series of gene fusions and completely synthetic genes modeled on the gene fusions.
This invention provides: 1) methods for the identification of broad-spectrum holins with a high level of nonenzymatic activity to breach microbial outer membranes and thereby increasing the efficacy of both natural plant defense compounds and artificially applied compounds; 2) conditions required for maintaining and increasing the anti-microbial and anti-pest efficacy of holins in gene fusions; 3) methods for effective targeting of holins expressed in plants through use of a xylem enhanced promoter and a leader peptide to direct the holin protein to the plant apoplast and xylem; 4) methods for the control of bacterial and fungal diseases of plants and control of insect and nematode pests that attack plants by expression of gene fusions involving holins, C-terminal additions and leader peptides, and optionally, endolysins; 5) methods for increasing the shelf-life of cut flowers; and 6) transgenic plants useful for the production of novel antimicrobial proteins based upon holins.